Over the last decade, biodegradable networks that can be formed in situ have been extensively researched. The advantages of using such networks compared to the conventional methods are localized delivery of active ingredient, cost effectiveness and patient compliance. Microspheres have been the choice for the delivery of drugs and proteins for long time. The major limitations of microspheres are reproducibility and the possibility of microsphere migration from the site of injection. The other disadvantage of microspheres is the number of steps involved and the need for use of solvents during synthesis of microspheres. The residual solvent is clearly undesirable during end application. Micelles have also been used in injectable drug delivery systems. However, along with the disadvantages of migration from the site of injection, the stability of micelles has been a concern. The stability of micelles depends on several variables such as critical micelle concentration, temperature, salt concentration and chemical structure of polymer. Any change in each of these variables can destabilize the micelle. In comparison to microspheres and micelles, the problem of migration of the delivery system can be eliminated by the use of an in situ formed hydrogel. The use of in situ formed hydrogel eliminates the washing and precipitation steps involved in the preparation and purification of microspheres, which can reduce the time and cost of making the product. The other distinctive advantage of in situ formed hydrogels compared to implants is the less invasive and painful insertion into the desired site. In comparison to drug delivery depot implants, the injectable hydrogels avoid the need for local anesthesia, surgical steps during implantation, post surgical complication and scars. The biodegradable hydrogels formed in situ have found application from tissue engineering (K. T. Nguyen, J. L. West, Biomaterials 23, 2002, 4307-4314) to cell release (T. Chen, D. A. Small, M. K. McDermott, W. E. Bentley, G. F. Payne, Biomacromolecules 4, 2003, 1558) and drug delivery system (A. Hatefi, B. Amsden, Journal of Controlled Release 80, 2002, 9-28).
Several methods are reported in the literature for the preparation of in situ crosslinked hydrogels, which can be broadly classified into two categories: 1. Physical crosslinking methods and 2. Chemical crosslinking methods. Physical crosslinking of hydrogels includes mechanisms such as ionic interaction, hydrophobic interaction and stereocomplexation. Ionic interaction has been used to prepare hydrogels by complexing cationic salt or polymer with anionic salt or polymer. Alginates are known to crosslink upon interaction with the ion such as calcium ion (C. P. Reis, A. J. Ribero, R. J. Neufeld, F. Veiga, Biotechnology and bioengineering, 96, 2007, 977-989). Wang et al (C. Wang, H. Liu, Q. GAO, X. Liu, Z. Tong, Carbohydrate Polymers 71, 2008, 476-480) have reported novel alginate/CaC03 hybrid hydrogels fabricated by in situ release of calcium ion by hydrolysis of D-glucano-δ-lactone to reduce pH. By controlling the alginate to CaC03 weight ratio, it was possible to tailor the mechanical strength of the hybrid hydrogels.
Amongst the physical crosslinking methods, the gels formed as a result of hydrophobic interactions have been extensively studied. The formation of the gel is a temperature dependent phenomenon. As the temperature is increased, hydrophobic association takes place which eventually leads to the formation of the gel from the sol. Research in this area has been focused on poly(ethylene glycol) or pluronic block with hydrophobic polylactide, PLGA or PTMC (S. Y. Kim, H. J. Kim, K. E. Lee, S. S. Han, Y. S. Sohn, B. Jeong, Macromolecules, 2007, 40 5519-5525; B. Jeong, Y. H. Bae, S. W. Kim Journal of Biomedical Materials Research 50, 2000, 171-177, Q. Hou, D. Y. S. Chau, C Pratoomsoot, P. J. Tighe, H. S. Dua, K. M. Shakesheff, F. R. A. J. Rose, Journal of Pharmaceutical Sciences, 2008, 1-9; Y. M. Kwon, S. W. Kim Macromolecular Symposia, 201, 2003, 179-186; B. Jeong, Y. H. Bae, S. W. Kim, Journal of Controlled Release, 63, 2000, 155-163, C. He, S. W. Kim, D. S. Lee, Journal of Controlled Release, 127, 3, 2008, 189-207). The gels formed at temperatures close to human body temperature, thus could be used for injectable drug delivery systems. Another approach to prepare temperature sensitive hydrogels is based on the polymer having lower critical solution temperature. Vermonden et al (T. Vermonden, N. A. M. Besseling, M. J. Van Steenbergen, W. E. Hennink, Langmuir 2006, 22, 10180) prepared triblock (ABA) copolymers containing thermosensitive poly(N-(2-hydroxy propyl)methacryl amide lactate as block A and hydrophilic poly(ethylene glycol) as block B. Triblock polymers gelled rapidly on increasing the temperature to body temperature, which makes them suitable for injectable drug delivery systems. In yet another approach, supramolecular complex of polymer with cyclodextrin has been used to prepare in situ gels. Li et al (J. Li, X. Ni, K. W. Leong, Journal of Biomedical Materials Research, 65A, 2003, 196-202) investigated hydrogels resulting from the supramolecular self assembly comprising poly(ethylene oxide) s and α-cyclodextrin. The rheological data showed that the gels were thixotropic and reversible. In another work, Manakker et al (F. van de Manakker, M. van der Pot, T Vermonden, C. F. van Nostrum, W. E. Hennink, Macromolecules 41, 2008, 1766-1773) prepared poly(ethylene glycol) hydrogel based on inclusion complexes between β-cyclodextrin and cholesterol. The rheological studies showed that the hydrogels were thermoreversible. Physical crosslinking through hydrophobic interactions can also be achieved by thermogelling the ionic polymer solutions such as chitosan solutions in the presence of counterionic salts such as β-glycerophosphate or ammonium hydrogen phosphate (E. Ruel-Gariepy, M. Shive, A. Bichara, M. Berrada, D. L. Garrec, A. Chemte, J. Leroux, European Journal of Pharmaceutics and Biopharmaceutics 57, 2004, 53-63; and L. S, Nair, T. Starnes, J K. Ko, C. T. Laurencin, Biomacromolecules 8, 2007, 3779-3785). The gelling time at body temperature varied from 5 min to 30 hours depending upon the concentration of ammonium hydrogen phosphate.
Two polymers containing complementary stereoregular groups when mixed together under appropriate conditions can form hydrogel. Aqueous solution of L-lactic acid oligomers and D-lactic acid oligomers grafted individually onto dextran, when mixed together at room temperature undergo gelation by stereocomplexation (S. R. Van Tomme, A. Mens, C. F. van Nostrum, and W. E. Hennink, Biomacromolecules, 9, 2008, 158-165; G. W. Bos, J. J. L. Jacobs, J. W. Koten, S. van Tomme, T. Veldhuis, C. F. van Nostrum, W. D. Otter, W. E. Hennink, European Journal of Pharmaceutical Sciences 21, 2004, 561-567). The properties of the hydrogel varied with solid content of the gel, degree of substitution of lactic acid oligomers and degree of polymerization of oligolactate graft. Hiemstra et al (C. Hiemstra, Z. Zhong, L. Li, P. J. Dijkstra, J. Feijen, Biomacromolecules 7, 2006, 2790-2795) synthesized stereo complexed hydrogels by mixing eight arm poly(ethylene glycol)-poly(L-lactate) and poly(ethylene glycol)-poly(D-lactate). The concentration of the polymer in the solution governed storage modulus and gelation time.
The two well known disadvantages of physically crosslinked hydrogels are that their mechanical properties are weaker compared to chemically crosslinked hydrogels. Further any change in external environment such as pH, temperature and ionic strength may disturb the inherent interaction, causing the disruption of the gel. To prepare hydrogels which do not revert to sol form in the external environment, a chemical crosslinking approach is needed. Chemical crosslinking methods include photocrosslinking, redox initiation, complementary group crosslinking and condensation reactions.
Photopolymerisation methods involve the irradiation of polymer solution with visible or UV light to form an in situ network in the presence of a photoinitiator. Polymers containing acrylate and methacrylate groups undergo rapid crosslinking upon irradiation with UV light. The system that has been largely explored using photopolymerisation technique is a triblock system, which consists of poly(ethylene glycol) block flanked with biodegradable oligo block bearing terminal acrylate functionality (K. S. Anseth, A. T. Metiers, S. J. Bryant, P. J. Martens, J. H. Elisseeff, C. N. Bowman, Journal of Controlled Release 78, 2002, 199-209; T. Matsuda, M. Mizutani, Journal of Biomedical Materials Research 62, 2002, 395-403). The biodegradable blocks used are oligo caprolactone, oligo lactide and oligo glycolide. The triblock containing terminal acrylate functionality, rapidly crosslinked under visible or UV light in the presence of photoinitiator. Degradation time of the gel varied with the molecular weight of the biodegradable component used. Recently, there have been attempts to prepare supramolecular complex of the triblock macromonomer having terminal double bond and alpha-cyclodextrin (S. Zhao, L. Zang, D. Ma, C. Yang, L. Yan Journal of Physical Chemistry B 110, 2006, 16503-16507; F. Zeng-guo, Z. Sanping, Polymer 44, 2003, 5177-5186). The inclusion complex was subsequently photopolymerised in the presence of photoinitiator to obtain a biodegradable hydrogel. The combination of physical crosslinking through supramolecular complex and chemical crosslinking through photocrosslinking led to a hydrogel having high mechanical strength. Wei and colleagues (H. Wei, H. Yu, A. Zhang, L. Sun, D. Hou, Z. Feng, Macromolecules 38, 2005, 8833-8839) prepared a thermosensitive, supramolecular structured hydrogel by copolymerization of N-isopropylacrylamide with alpha-cyclodextrin threaded amphiphilic LA-PEG-LA copolymer end capped with a methacrylate group. The thermosensitivity of these hydrogels was modulated by varying the N-isopropyl-acrylamide content as well as the alpha-cyclodextrin to amphiphilic macromer ratio. In another attempt, oligomeric poly(2-hydroxyethylmethacrylate) and poly(N,N,-dimethyl acrylamide) were grafted onto hyaluronic acid followed by modification with glycidyl methacrylate (X. Jia, J. A. Burdick, J. Kobler, R. J. Clifton, J. J. Rosowski, S. M. Zeitels, R. Langer, Macromolecules 37, 2004, 3239-3248). The aqueous solution of macromonomer was photopolymerised in the presence of photoinitiator to obtain the gel. When compared with hyaluronic acid reacted with glycidyl methacrylate, the modified gel was resistant to enzymatic degradation and lower swelling. Doulabi et al (A. S. H. Doulabi, H. Mirzadeh, M. Imani, S. Sharifi, M. Atai, S. Mehdipour-Ataie, Polymers In Advanced Technologies, 2008) reported in situ formed devices based on biodegradable macromer comprising poly(ethylene glycol) and fumaric acid copolymers. The macromers were photocrosslinked for 300 seconds in the presence of visible light, initiator, accelerator and a reactive diluent. The use of N-vinylpyrrolidone as a reactive diluent increased the crosslink density and shrinkage strain.
The other method to prepare chemically crosslinked hydrogel at room temperature is by free radical polymerization using redox initiators. Franssen et al (O. Franssen, R. D. van Ooijen, D. de Boer, R. A. A. Maes, W. E. Hennink, Macromolecules 32, 1999, 2896-2902) synthesized dextran methacrylate based gel with redox initiators such as N,N,N,N-tetramethylethylenediamine and potassium peroxodisulfate. The aqueous solution of initiator and dextran methacrylate formed gel overnight at room temperature. The enzymatic degradability of the gel was studied with dextranase. Holland et al (T. A. Holland, J. K. V. Tessmar, Y. Tabata, A. G. Mikos Journal of controlled release 94, 2004, 101-114) studied the degradation and TGF release from gelatin microparticles embedded in biodegradable oligo-poly(ethylene glycol) fumarate network. The crosslinked hydrogel was prepared by reacting oligo-poly(ethylene glycol) fumarate with methylene biscrylamide using redox initiators tetramethylethylene diamine and ammonium persulfate. The hydrogel formed in 10 minutes at 37° C. An intelligent and biodegradable hydrogel was prepared by crosslinking temperature sensitive triblock poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) with extension along each ends with oligo caprolactone terminated with acrylate group (B. Wang, W. Zhu, Y. Zhang, Z. Yang, J. Ding, Reactive and Functional Polymers 66, 2006, 509-518). The acrylate terminated macromer was dissolved in water and crosslinked using redox initiators tetramethylethylene diamine and ammonium persulfate. The hydrogel showed reversible temperature sensitive swelling/deswelling characteristics. Other groups have prepared hydrogels by chemical crosslinking of functional groups by condensation of a water soluble polymer with appropriate reactive reagents such as diisocyanates, glutaraldehyde and carbodiimides (U.S. Pat. No. 5,078,744; Tomihata, K, Ikada, Y., Journal of Polymer Science, Part A: Polymer Chemistry 35, 1997, 3553-3559; Tomihata, K; Ikada, Y., Journal of Biomedical Materials Research 37, 1997, 243-251). Eiselt et al (P. Eiselt, K. Y. Lee, D. J. Mooney, Macromolecules 32, 1999, 5561-5566) prepared alginate hydrogels with poly(ethylene glycol)diamine using l-ethyl-3-(dimethyl aminopropyl)carbodiimide (EDC). Hydrogels of alginate with poly(ethylene glycol)diamines of different molecular weights were investigated to generate materials with a range of mechanical properties. However, these chemically crosslinked networks suffer from various limitations, such as the use of photoirradiation, the presence of residues of initiators, organic solvents and other reagents. The presence of such impurities is generally not acceptable in biomedical applications.
In order to overcome above mentioned limitations, chemical crosslinking of complementary groups has been studied. Recently, hydrogels formed from the chemical reaction of low molecular weight amines with N-hydroxysuccinimide based crosslinker groups was reported (Pub. No. US2009/0324721). However the limitations of this system include the loss of reactivity of functional groups during reaction and storage due to hydrolysis, incorporation of extended biodegradable region (extra synthetic engineering required) and the use of low molecular weight amines (high probability of leaching out to the surrounding during crosslinking). Another way of preparing hydrogel using chemical crossing of complementary groups is the reaction of thiol with vinyl moieties. Hiemstra et al (C. Hiemstra, L. J. van der Aa, Z. Zhong, P. J. Dijkstra, J. Feijen, Macromolecules 40, 2007, 1 165-1 173; C. Hiemstra, Z. Zhong, M. J. van Steenbergen, W. E. Hinnink, J. Feijen, Journal of Controlled Release 122, 2007, 71-78) synthesized various vinylsulfone containing dextrans and reacted with multi functional poly(ethylene glycol) having thiol group. Hydrogel formed rapidly upon mixing in aqueous medium under physiological condition. The degradation time of the gel varied from 3 to 21 days. Besides dextran, hyaluronic acid has also been reported to form in situ gels. Shu et al (X. Z. Shu, Y. Liu, F. S. Palumbo, Y. Luo, G. D. Prestwich, Biomaterials 25, 2004, 1339-1348) reported two thiolated hyaluronan derivatives coupled to four α,β-unsaturated esters and amides. It was disclosed that the gelation time varied from 10 minutes to 2 hrs. Subsequently, Vanderhooft et al (J. L. Vanderhooft, B. K. Mann, G. D. Prestwich, Biomacromolecules 8, 2007, 2883-2889) synthesized novel thiol reactive poly(ethylene glycol) crosslinkers to prepare hydrogel with thiol mediated derivative of hyaluronan. The gelation time at physiological pH varied from 1 minute to over 2 hrs. Hydrogel mimics of extracellular matrix were synthesized by crosslinking thiol modified analog of heparin and thiol modified hyaluronan with polyethylene glycol diacrylate (S. Cai, Y. Liu, X. Z. Shu, G. D. Prestwich Biomaterials 26, 2005, 6054-6067). The water content of the hydrogel was more than 97% and gelation occurred within 10 minutes. Similarly, hyaluronic acid based gels were prepared by Kim et al (G. Kim, Y. Choi, M. Kim, Y. Park, K. Lee, I. Kim, S. Hwang, I. Noh, Current Applied Physics 7SI, 2007, e28-e32) using aminopropylmethacrylate modified hyaluronic acid and multifunctional poly(ethylene glycol) with thiol groups. Thiol functionalization was also utilized to prepare poly(vinyl alcohol) based hydrogel for in vitro retinal replacement surgery (M. Tortora, F. Cavalieri, E. Chiessi, G. Paradossi Biomacromolecules 8, 2007, 209-214). The hydrogel was formed by the reaction of thio capped poly(vinyl alcohol) with methacrylate derivative of poly(vinyl alcohol). Poly(vinyl alcohol) hydrogel gelled in 60 minutes at physiological condition in aqueous medium. Qiu et al (B Qiu, S. Stefanos, J. Ma, A. Lalloo, B. A. Perry, M. J. Leibowitz, P. J. Sinko, S. Stein, Biomaterials 24, 2003, 11-18) described a hydrogel from poly(ethylene glycol) based copolymer with multifunctional thiol and divinyl sulfone poly(ethylene glycol). The aqueous mixture of the composition formed a gel in 2-3 minutes. A major disadvantage of the system is the fate of thiol group during storage and application, as thiol groups are known to be oxidized to disulfide by oxygen. Moreover thiol compounds are known to degrade the disulfide bond present in the protein, leading to denaturation. Again the variation in degradation time of the gel is brought about by sacrificing the crosslinking density.
References may be made to an article “D. M. Lynn, D. G. Anderson, D. Putnam, R. Langer, Journal of the American Chemical Society 123, 2001, 8155-8156” wherein the synthesis of poly(β-aminoester) by the addition of amine to diacrylate ester has been described. Further, WO2004/106411 discloses a method for synthesizing poly(beta-amino esters) prepared from the addition of bis(secondary amines) or primary amines with bis(acrylate ester) in an organic solvent, wherein the amine is in the form of polynucleotide/polymer complexes (DNA/polymer concentration) and polyethylene glycol polymers. U.S. Pat. No. 6,998,115 discloses a method for preparation of the polymers, specifically the poly β-amino esters and salts which are useful for the drug delivery devices and pharmaceutical composition. The poly β-amino esters are prepared by the conjugate addition of bis(secondary amines) or primary amines to bis(acrylate ester). The monomers are dissolved in an organic solvent such as methylene chloride and the resulting solution is further combined and heated for the polymerization of the said monomers. The polymerization time was 5 days at 45° C.
References may be made to patent WO2008/011561, wherein end-modified poly(beta-amino esters) prepared by the addition of a nucleophilic reagent (e.g., an amine) to an acrylate-terminated poly(beta-amino ester) which are useful in a variety of medical applications including drug delivery, tissue engineering, and biomaterials and non-medical applications including coatings, plastics, paints, and films are disclosed. Further, the invention discloses a method of synthesizing an end-modified poly(β-amino ester), comprising following steps: a) providing an amine-terminated poly(β-amino ester); b) providing an acrylate; and c) reacting the acrylate and the amine-terminated poly(β-amino ester) under suitable conditions to form an end-modified poly(β-amino ester). The polymerization time was typically 24 hrs at 90° C. for the preparation of acrylate modified poly(β-amino esters).
Recently, a combinatorial library of photocrosslinkable and degradable poly(β-aminoester) was synthesized and evaluated (D. G. Anderson, C. A. Tweedie, N. Hossain, S. M. Navarro, D. M. Brey, K. J. Van Vliet, R. Langer, J. A. Burdick, Advanced Materials 18, 2006, 2614-2618). Macromers of different molecular weight and chemical structures bearing a terminal acrylate group were synthesized by the reaction of excess diacrylates with primary or bis(secondary) amines. The macromers were photopolymerised with UV lamp in the presence of photoinitiator 2,2-dimethoxy-2-phenylacetophenone dissolved in 10 wt % of dichloromethane. The library of 120 poly(β-aminoester) (PBAE) networks exhibited a wide range of degradation behaviour with mass loss of 100% within 24 hrs to little mass loss even after 57 days of immersion. These variations were brought about by simple variation in diacrylate and amines used. Brey et al (D. M. Brey, J. L. Ifkovits, R. I. Mozia, J. S. Katz, J. A. Burdick, Acta Biomaterialia 4, 2008, 207-217) disclosed a large library of PBAEs, synthesized via a step growth polymerization of liquid amines and diacrylates developed for biomaterial applications. The said article also disclosed and synthesized a macromer system, with a variety of branching structures, to illustrate the diverse properties possible through this structural variation. The system involves the synthesis, from a diacrylate (E) and a primary amine to form a linear diacrylated macromer, or alternatively, a system of branched multiacrylated and linear diacrylated macromers with the addition of small quantities of triacrylate (PETA). The macromers were dissolved in methylene chloride and photopolymerised in the presence of a photoinitiator 2,2-dimethoxy-2-phenylacetophenone to obtain poly(β-amino ester) networks. The increase in macromer branching improved the gel content and mechanical properties of the networks.
References may be made to journal “T. Kim, H. J. Seo, J. S. Choi, J. K. Yoon, J. Back, K. Kim, J. Park, Bioconjugate Chemistry 16, 2005, 1 140-1 148” and “J. Kloeckner, E. Wagner, M. Ogris, European Journal of Pharmaceutical Sciences 29, 2006, 414-425” wherein biodegradable crosslinked poly(β-aminoester) synthesized directly by conjugate addition of diacrylates with amines in different organic solvents at 50-60° C. for 2-4 days has been reported.
The current strategies for the synthesis of crosslinked poly(β-aminoester) networks include use of photo polymerization and/or thermal curing. However, these crosslinked networks suffer from many limitations, such as the use of photoirradiation, the presence of residual initiators, organic solvents, other reagents, high reaction temperature and high reaction times. After conducting several laboratory scale methods it is surprisingly found that aqueous solutions containing water soluble diacrylates and aqueous solutions containing multifunctional amines, when mixed together react rapidly and yield hydrogels. Since, the gelation temperature is close to human body temperature, the technique can be used to synthesize in situ formed hydrogels which further degrade under physiological conditions.